H. pylori is a Gram negative bacterium that colonizes the human gastric mucosa causing chronic gastritis that may progress to peptic ulceration and gastric cancer (Blaser, M. J., and D. E. Berg. 2001. Helicobacter pylori genetic diversity and risk of human disease. J Clin Invest 107:767-773). H. pylori expresses adhesins on its surface which provides intimate adherence to the gastric mucosa, allowing these auxotroph organisms to gain nutrients from host tissues. The BabA adhesin is a member of the paralogous family of outer membrane proteins (Alm, R. A., J. Bina, B. M. Andrews, P. Doig, R. E. Hancock, and T. J. Trust. 2000. Comparative genomics of Helicobacter pylori: analysis of the outer membrane protein families. Infect Immun 68:4155-4168) that binds to fucosylated ABO/Lewis b blood group antigens which are expressed on epithelial cells (Boren, T., P. Falk, K. A. Roth, G. Larson, and S. Normark. 1993. Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. Science 262:1892-1895 and Ilver, D., A. Arnqvist, J. Ogren, I. M. Frick, D. Kersulyte, E. T. Incecik, D. E. Berg, A. Covacci, L. Engstrand, and T. Boren. 1998. Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging. Science 279:373-37).
BabA is a possible vaccine candidate since a high proportion of clinical isolates has been shown to express BabA (Ilver, D., A. Arnqvist, J. Ogren, I. M. Frick, D. Kersulyte, E. T. Incecik, D. E. Berg, A. Covacci, L. Engstrand, and T. Boren. 1998. Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging. Science 279:373-377; Kim, S. Y., C. W. Woo, Y. M. Lee, B. R. Son, J. W. Kim, H. B. Chae, S. J. Youn, and S. M. Park. 2001. Genotyping CagA, VacA subtype, IceA1, and BabA of Helicobacter pylori isolates from Korean patients, and their association with gastroduodenal diseases. J Korean Med Sci 16:579-584, and Mizushima, T., T. Sugiyama, Y. Komatsu, J. Ishizuka, M. Kato, and M. Asaka. 2001. Clinical relevance of the babA2 genotype of Helicobacter pylori in Japanese clinical isolates. J Clin Microbiol 39:2463-2465). Active vaccination against H. pylori is difficult, due to the low immune activity in the GI-tract. One other issue is the high recombination rate of H. pylori. Passive immunization on the contrary is more advantageous, because the Abba3 antibodies bind to the antigen, making it difficult for the H. pylori to adhere to the mucosa.
Recent studies have also demonstrated a significant association between the expression of BabA and development of peptic ulcer and gastric cancer (Gerhard, M., N. Lehn, N. Neumayer, T. Boren, R. Rad, W. Schepp, S. Miehlke, M. Classen, and C. Prinz. 1999. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin. Proc Natl Acad Sci USA 96:12778-12783 and Prinz, C., N. Hafsi, and P. Voland. 2003. Helicobacter pylori virulence factors and the host immune response: implications for therapeutic vaccination. Trends Microbiol 11:134-138). Currently, the feasibility of passive immunotherapy by delivery of highly ABO/Lewis b fucosylated glycoconjugates is being investigated (Gustafsson, A., A. Hultberg, R. Sjostrom, I. Kacskovics, M. E. Breimer, T. Boren, L. Hammarstrom, and J. Holgersson. 2006. Carbohydrate-dependent inhibition of Helicobacter pylori colonization using porcine milk. Glycobiology 16:1-10., and Xu, H. T., Y. F. Zhao, Z. X. Lian, B. L. Fan, Z. H. Zhao, S. Y. Yu, Y. P. Dai, L. L. Wang, H. L. Niu, N. Li, L. Hammarstrom, T. Boren, and R. Sjostrom. 2004. Effects of fucosylated milk of goat and mouse on Helicobacter pylori binding to Lewis b antigen. World J Gastroenterol 10:2063-2066). Alternatively, antibody derivatives that interfere with mucosal adherence could be administered orally or produced in situ by GRAS (Generally Recognised As Safe) microorganisms as shown by oral administration of IgG against H. pylori (Casswall, T. H., H. O, Nilsson, L. Bjorck, S. Sjostedt, L. Xu, C. K. Nord, T. Boren, T. Wadstrom, and L. Hammarstrom. 2002. Bovine anti-Helicobacter pylori antibodies for oral immunotherapy. Scand J Gastroenterol 37:1380-1385, and Keenan, J., S, Neal, R. Allardyce, and J. Roake. 2002. Serum-derived IgG1-mediated immune exclusion as a mechanism of protection against H. pylori infection. Vaccine 20:2981-2988), or by the administration of a Lactobacillus expressing a single-chain variable fragment (scFv) against Streptococcus mutants (Kruger, C., Y. Hu, Q. Pan, H. Marcotte, A. Hultberg, D. Delwar, P. J. Van Dalen, P. H. Pouwels, R. J. Leer, C. G. Kelly, C. van Dollenweerd, J. K. Ma, and L. Hammarstrom. 2002. In situ delivery of passive immunity by lactobacilli producing single-chain antibodies. Nat Biotechnol 20:702-706). scFv is a genetically engineered antibody that consists of the variable heavy chain (VH) and light chain (VL) of an immunoglobulin joined together by a flexible peptide linker.
The bacteria in the stomach are not only confronted by host-specific environmental conditions but also face changes of the mucosal glycosylation pattern during disease progression. The remarkable ability of H. pylori to establish a chronic and persistent infection despite is likely due to be due its extraordinarily high recombination rate (Falush, D., C. Kraft, N. S. Taylor, P. Correa, J. G. Fox, M. Achtman, and S. Suerbaum. 2001. Recombination and mutation during long-term gastric colonization by Helicobacter pylori: estimates of clock rates, recombination size, and minimal age. Proc Natl Acad Sci USA 98:15056-15061).
The ability of H. pylori to switch between tight adherence and non-adherence gives the bacterium access to nutrients leaking from the inflamed tissue but also exposes the bacterium to the inflammatory host response (Rhen, M., S. Eriksson, M. Clements, S. Bergstrom, and S. J. Normark. 2003. The basis of persistent bacterial infections. Trends Microbiol 11:80-86).
BabA contributes to the flexibility in binding by frameshift-based variation in the CT-rich leader-sequence, horizontal gene transfer and gene conversion with babB and babC (Aspholm-Hurtig, M., G. Dailide, M. Lahmann, A. Kalia, D. Ilver, N. Roche, S. Vikstrom, R. Sjostrom, S. Linden, A. Backstrom, C. Lundberg, A. Arnqvist, J. Mandavi, U. J. Nilsson, B. Velapatino, R. H. Gilman, M. Gerhard, T. Alarcon, M. Lopez-Brea, T. Nakazawa, J. G. Fox, P. Correa, M. G. Dominguez-Bello, G. I. Perez-Perez, M. J. Blaser, S, Normark, I. Carlstedt, S. Oscarson, S. Teneberg, D. E. Berg, and T. Boren. 2004. Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin. Science 305:519-522; Backstrom, A., C. Lundberg, D. Kersulyte, D. E. Berg, T. Boren, and A. Arnqvist. 2004. Metastability of Helicobacter pylori bab adhesin genes and dynamics in Lewis b antigen binding. Proc Natl Acad Sci USA 101:16923-16928 and Solnick, J. V., L. M. Hansen, N. R. Salama, J. K. Boonjakuakul, and M. Syvanen. 2004. Modification of Helicobacter pylori outer membrane protein expression during experimental infection of rhesus macaques. Proc Natl Acad Sci USA 101:2106-2111), outer membrane proteins (OMPs) closely related to BabA in their N- and C-terminal region but located in different loci. Even though BabA is mainly found in the babA locus, gene conversion with BabB located in the babB locus leads to the formation of either full a length BabA under the control of the weaker BabB promoter or to chimeric BabA/BabB genes with the site of recombination upstream of the unique region that distinguishes BabA from BabB (Colbeck, J. C., L. M. Hansen, J. M. Fong, and J. V. Solnick. 2006. Genotypic profile of the outer membrane proteins. BabA and BabB in clinical isolates of Helicobacter pylori. Infect Immun 74:4375-4378. 13. Dubel, S., F. Breitling, P. Fuchs, M. Braunagel, I. Klewinghaus, and M. Little. 1993. A family of vectors for surface display and production of antibodies. Gene 128:97-101). Recombination can also lead to the presence of BabB within the babA locus with subsequent loss of Lewis b binding in infected macaques and patients (Colbeck, J. C., L. M. Hansen, J. M. Fong, and J. V. Solnick. 2006. Genotypic profile of the outer membrane proteins BabA and BabB in clinical isolates of Helicobacter pylori. Infect Immun 74:4375-4378; Dubel, S., F. Breitling, P. Fuchs, M. Braunagel, I. Klewinghaus, and M. Little. 1993. A family of vectors for surface display and production of antibodies. Gene 128:97-101; Hennig, E. E., J. M. Allen, and T. L. Cover. 2006. Multiple chromosomal loci for the babA gene in Helicobacter pylori. Infect Immun 74:3046-3051; Hennig, E. E., R. Mernaugh, J. Edl, P. Cao, and T. L. Cover. 2004. Heterogeneity among Helicobacter pylori strains in expression of the outer membrane protein BabA. Infect Immun 72:3429-3435 and Solnick, J. V., L. M. Hansen, N. R. Salama, J. K. Boonjakuakul, and M. Syvanen. 2004. Modification of Helicobacter pylori outer membrane protein expression during experimental infection of rhesus macaques. Proc Natl Acad Sci USA 101:2106-2111). The third locus BabC, formerly only described in strain 26695, has recently been shown to be involved in the recombination exchange with BabA in additional strains (Colbeck, J. C., L. M. Hansen, J. M. Fong, and J. V. Solnick. 2006. Genotypic profile of the outer membrane proteins BabA and BabB in clinical isolates of Helicobacter pylori. Infect Immun 74:4375-4378. 13. Dubel, S., F. Breitling, P. Fuchs, M. Braunagel, I. Klewinghaus, and M. Little. 1993. A family of vectors for surface display and production of antibodies. Gene 128:97-101; Hennig, E. E., J. M. Allen, and T. L. Cover. 2006. Multiple chromosomal loci for the babA gene in Helicobacter pylori. Infect Immun 74:3046-3051. 21. Hennig, E. E., R. Mernaugh, J. Edl, P. Cao, and T. L. Cover. 2004. Heterogeneity among Helicobacter pylori strains in expression of the outer membrane protein BabA. Infect Immun 72:3429-3435).
Backström et al. (Backstrom, A., C. Lundberg, D. Kersulyte, D. E. Berg, T. Boren, and A. Arnqvist. 2004. Metastability of Helicobacter pylori bab adhesin genes and dynamics in Lewis b antigen binding. Proc Natl Acad Sci USA 101:16923-16928) have shown that among clinical H. pylori isolates which had lost their ability to bind Lewis b, a small fraction of the bacterial population harbored a BabB/BabA chimera and Lewis b binding could be reconstituted by panning with Lewis b coated magnetic beads in vitro. This will enable the bacterium to respond to a changing glycosylation pattern upon an inflammatory host response. Gene conversion leads to the formation of a mosaic pattern of BabA, but mutation of the CBD (carbohydrate-binding domain) will lead to the loss of function and thereby to a reduced survival of H. pylori in the acute phase of infection in which the blood group antigens are still highly expressed on the epithelial cells.
The exclusive presence of babB in the babA locus of analyzed strains at the end of an experimental infection of rhesus macaques has led to the hypothesis that antigenic variation is used to avoid the host immune response (Solnick, J. V., L. M. Hansen, N. R. Salama, J. K. Boonjakuakul, and M. Syvanen. 2004. Modification of Helicobacter pylori outer membrane protein expression during experimental infection of rhesus macaques. Proc Natl Acad Sci USA 101:2106-2111). The antigenicity of H. pylori antigens in patient's sera has been tested in two reports but since the proteins were denatured with urea prior to 2D-separation, the possibility to detect conformational dependent membrane proteins was rather limited (Haas, G., G. Karaali, K. Ebermayer, W. G. Metzger, S. Lamer, U. Zimny-Arndt, S. Diescher, U. B. Goebel, K. Vogt, A. B. Roznowski, B. J. Wiedenmann, T. F. Meyer, T. Aebischer, and P. R. Jungblut. 2002. Immunoproteomics of Helicobacter pylori infection and relation to gastric disease. Proteomics 2:313-324 and Kimmel, B., A. Bosserhoff, R. Frank, R. Gross, W. Goebel, and D. Beier. 2000. Identification of immunodominant antigens from Helicobacter pylori and evaluation of their reactivities with sera from patients with different gastroduodenal pathologies. Infect Immun 68:915-920).
Polyclonal anti-BabA sera have been raised by immunization of rabbits with recombinant BabA isolated from inclusion bodies (Yamaoka, Y., J. Souchek, S. Odenbreit, R. Haas, A. Arnqvist, T. Boren, T. Kodama, M. S. Osato, O. Gutierrez, J. G. Kim, and D. Y. Graham. 2002. Discrimination between cases of duodenal ulcer and gastritis on the basis of putative virulence factors of Helicobacter pylori. J Clin Microbiol 40:2244-2246) and shown to recognize BabA in a majority of strains. To date, two monoclonal BabA specific scFvs have been described which were generated by immunization of rodents with a BabA-GST fusion protein, covering the BabA-J99 domain from amino acid 128 to 310 (Hennig, E. E., R. Mernaugh, J. Edl, P. Cao, and T. L. Cover. 2004. Heterogeneity among Helicobacter pylori strains in expression of the outer membrane protein BabA. Infect Immun 72:3429-3435). Only about half of the analyzed strains were positive by Westernblot analysis, probably due to the more restricted epitope of a monoclonal antibody in comparison to a polyclonal serum. In addition, the phage selection of the described scFv was performed on recombinant protein containing only a portion of BabA with no defined functional conformation.
A new approach in preventing infectious diseases transmitted through mucosal sites consists of the in situ delivery of antibody fragments by lactobacilli or other GRAS microorganisms (Kruger, C., Y. Hu, Q. Pan, H. Marcotte, A. Hultberg, D. Delwar, P. J. Van Dalen, P. H. Pouwels, R. J. Leer, C. G. Kelly, C. van Dollenweerd, J. K. Ma, and L. Hammarstrom. 2002. In situ delivery of passive immunity by lactobacilli producing single-chain antibodies. Nat Biotechnol 20:702-706).
The BabA adhesin has previously been identified and shown to be localized on the bacterial surface of H. pylori (SE 9602287-6). The blood group binding activity was shown to be pH dependent and the present inventors present evidence that the binding affinity to the Lewis-b receptor reveals a high equilibrium constant.
Intensive research has been directed to the immunological treatment and prevention of H. pylori induced infections. EP 0 484 148 (Ando & Nakamura) describes a method for treating and/or preventing upper gastrointestinal disease in mammals, said method comprising orally administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising anti-H. pylori polyclonal immunoglobulins and a pharmaceutically acceptable carrier. The description further dwells on the combination of said treatment in combination with the administration of antibiotics. As the method of producing said polyclonal antibodies, EP 0 484 148 describes the isolation and purification of anti-H. pylori immunoglobulins from the sera and milk of mammals. H. pylori itself was not found in the stomachs of cows, goats, sheep, swine or horses, according to EP 0 484 148, but it was assumed that these animal species have colonizing microorganisms with antigenic determinants similar to those of H. pylori because they have immunoglobulins which cross-react to strains of H. pylori found in humans. Preferably, according to EP 0 484 148, large mammals, e.g. pregnant cows, are immunized with whole cells of H. pylori and the immunoglobulins subsequently extracted from the milk or colostrum. In the immunization experiments, NCTC Strain 11362 and clinical isolate H. pylori No. 153 were used to trigger the production of immunoglobulins. On the other hand, NCTC Strain 11637 was used for analyzing purposes. Immunization is claimed to yield an anti-H. pylori titer in the milk of such magnitude, that daily doses of 0.01-0.1 g/day immunoglobulin composition, are sufficient for successful therapy. The claimed interval of 0.01-0.1 g/day is however not supported by the experiments presented by Ando & Nakamura and so low doses have hitherto not proven efficient in clinical tests. The doses actually used in example 5 and 7 are in the order of magnitude of 1 g/day, i.e. 10-fold the upper limit of the given interval. Furthermore, it is very unlikely that unspecific immunoglobulin mixtures as those manufactured by Ando & Nakamura would be effective in claimed doses as similar doses are ineffective against other gastrointestinal pathogens. The simultaneous administration of antibiotics, extensively discussed in the description, underlines the insufficiency of the disclosed immunoglobulins.
EP 0 469 359 (Cordle & Schaller) likewise describes the immunization of mammals, preferably pregnant cows, with formalin killed H. pylori bacteria (ATCC Strain 26695). Anti-H. pylori polyclonal antibodies were isolated and purified from the milk and finally fed to piglets, in amounts of about 0.5 g immunoglobulins, three times daily. The results were assessed by determination of the number of biopsy specimens, which were positive for Gram-negative bacteria after the trial. Gram-negative bacteria were found in 78% of the piglets fed a non-immune nutrient but only (Sic!) in 35% of the piglets fed a nutrient containing so called specific anti-H. pylori antibodies.
US20040234529, a patent application of the same inventors as in the invention herein, discloses the BabA protein. Said adhesins and/or DNA are useful for diagnosis and therapy and/or prophylaxis directed against H. pylori induced infections, e.g. gastritis and acid peptic disease, i.e. active vaccination. They also disclose an immunoglobulin composition, which exhibits specific activity to a Lewis b antigen binding Helicobacter pylori adhesion for treatment and/or prevention of gastrointestinal diseases, caused by H. pylori for passive vaccination. Unlike the invention herein the antibody is an animal antibody and not a specific selected human antibody. Furthermore the applicants of the US20040234529 do not mention detection in fecal samples.
Even though the central part of BabA is most heterogeneous and determines the specificity of receptor binding (Aspholm-Hurtig, M., G. Dailide, M. Lahmann, A. Kalia, D. Ilver, N Roche, S. Vikstrom, R. Sjostrom, S. Linden, A. Backstrom, C. Lundberg, A. Arnqvist, J. Mandavi, U. J. Nilsson, B. Velapatino, R. H. Gilman, M. Gerhard, T. Alarcon, M. Lopez-Brea, T. Nakazawa, J. G. Fox, P. Correa, M. G. Dominguez-Bello, G. I. Perez-Perez, M. J. Blaser, S, Normark, I. Carlstedt, S. Oscarson, S. Teneberg, D. E. Berg, and T. Boren. 2004. Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin. Science 305:519-522 and Ilver, D., A. Arnqvist, J. Ogren, I. M. Frick, D. Kersulyte, E. T. Incecik, D. E. Berg, A. Covacci, L. Engstrand, and T. Boren. 1998. Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging. Science 279:373-377), the yet unmapped carbohydrate-binding domain (CBD) cannot be subjected to a high rate of mutation without loss of function; nevertheless fine-tuning during evolution was shown by adaptation of BabA in H. pylori strains isolated from indigenous South American population that preferentially bind to O-Lewis b, which is the predominant blood group antigen in this continent (nominated as “Specialists” strains). Contrarily, strains isolated from continents in which the A/B Lewis b blood group antigens are more evenly represented in the host population demonstrate a more general binding capability against A/B Lewis b (including O-Lewis b) and were hence named “Generalist” binder (Aspholm-Hurtig, M., G. Dailide, M. Lahmann, A. Kalia, D. Ilver, N. Roche, S. Vikstrom, R. Sjostrom, S. Linden, A. Backstrom, C. Lundberg, A. Arnqvist, J. Mandavi, U. J. Nilsson, B. Velapatino, R. H. Gilman, M. Gerhard, T. Alarcon, M. Lopez-Brea, T. Nakazawa, J. G. Fox, P. Correa, M. G. Dominguez-Bello, G. I. Perez-Perez, M. J. Blaser, S, Normark, I. Carlstedt, S. Oscarson, S. Teneberg, D. E. Berg, and T. Boren. 2004. Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin. Science 305:519-522). The BabA sequences were aligned but no specific babA domains that would correspond to Generalist versus Specialists could be mapped. We therefore aimed to develop an antibody with specificity for the receptor-binding site and used the phage display technique to enrich for antibodies from patients whose sera featured competitive BabA binding characteristics towards Lewis b.
It is already known in the art how to produce human scFv-libraries derived from peripheral blood lymphocytes against various peptides. It is also known how to select for denaturated, linear, non-native peptides of H. pylori. But no previous documents describe the selection method, selecting for the native, non-denaturated, three-dimensional BabA polypeptide.
Here, we present a human scFv-library derived from peripheral blood lymphocytes of H. pylori infected patients and one of the identified and selected BabA-specific human single chains was converted to a human IgG1 antibody, named Abba3. Abba3 scFv refers to the variable binding regions and includes derivatives thereof. Surprisingly the antibody binds to a majority of H. pylori clinical isolates and demonstrated similar binding characteristics as the A-Lewis b or B-Lewis b blood group sugar antigens, i.e. preferential recognition of the “Generalist” type of BabA, distributed most commonly world-wide. Abba3 antibodies neutralize the H. pylori by binding to BabA, making it difficult for the bacteria to adhere to the mucosa. Consequently the bacteria/antibody-complex disappears naturally from the GI-system. Since there is a low immunoactivity in the stomach-tract, antibody detection is less useful than antigen detection. Accordingly, a detection kit using Abba3 antibodies for detection of H. pylori in faecal samples are preferred. Because Abba3 is a fully human IgG1 antibody it has the advantage of being effective in the activation of complement-directed lysis of the bacteria, accordingly, also activating the immune system.
It is therefore an object of this invention to provide the antibody Abba3 selected for its ability to specifically bind to BabA of H. pylori, for use in passive vaccination. It is also an object of the invention to use Abba3 for antigen detection of H. pylori in faecal samples. Furthermore an object of this invention is to provide any antibody selected using the method described in the invention herein for its ability to specifically bind to BabA of H. pylori. Other objects and advantages will be more fully apparent from the following disclosure and appended claims.